Bibliographic details of the publications numerically referred to in this specification are collected at the end of the description.
A variety of nucleic acid amplification technologies exist for the diagnosis of infectious and genetic diseases. Since its invention over a decade ago, the polymerase chain reaction (PCR) (1) has become the method of choice in research and DNA-based diagnostics. This can be attributed to its speed, simplicity and sensitivity. PCR does, however, require temperature cycling, which therefore necessitates the use of expensive thermal cycling equipment. Other amplification techniques, which also require temperature cycling, include the ligase chain reaction (LCR) (2) and the transcription-based amplification system (TAS) (3).
Various other amplification techniques exist which do not require extensive thermal cycling and are essentially isothermal systems. Several of these are transcription-mediated or require RNA as an integral component of the reaction therefore necessitating that the amplification environment is kept free from ribonuclease contamination. These methods include the Qβ replicase system (4), self-sustained sequence replication (3SR) (5) and nucleic acid sequence-based amplification (NASBA) (6).
Presently, there appear to exist at least two isothermal techniques for the amplification of nucleic acid sequences which essentially do not require RNA intermediates. Strand displacement amplification (SDA) (7) is an isothermal technique which relies on the ability of a restriction enzyme to nick a hemiphosphorothioated recognition site and the ability of a polymerase to initiate replication at a nick and displace the downstream strand. The other isothermal technique which can be used to amplify a nucleic acid sequence is rolling circle amplification (RCA).
Various forms of the rolling circle amplification technique have previously been described (8, 9). In essence the technique relies on amplification from a circular DNA probe. The circular probe, commonly referred to as a “padlock probe”, is designed such that it has regions at both its 5′ and 3′ ends which are complementary to the target sequence of interest and are separated by a region of nucleotides of non-target derived origin. Upon hybridisation, the 5′ and 3′ ends of the probe are brought into close proximity to one another. If the two probe regions are adjacent to one another the 5′ and 3′ ends can be joined to produce a circular probe. In some instances, however, the probe regions are separated from one another by a small stretch of nucleotides. This region must be filled to achieve the generation of a circular probe. In this regard, a variety of techniques can be utilised including the use of spacer oligonucleotides or by using a DNA polymerase (or a reverse transcriptase in the case of an RNA target) in combination with deoxynucleotide triphosphate molecules to fill the gap prior to ligation.
A significant problem associated with the rolling circle amplification technique is the occurrence of background amplification. Prior to the advent of the present invention this background amplification was dismissed as primer-induced deletion fragment repeats encompassing a full unit repeat minus the intervening region between 5′ ends of the two primers (8). Background amplification represents both a significant problem and a limitation for rolling circle amplification reactions which utilise 2 primers. It is also a major source of false positive results. In fact, the magnitude of the problem presented by the occurrence of this background amplification has been such that it has not been feasible to use the two primer rolling circle amplification techniques with an acceptable level of specificity.
In work leading up to the present invention the inventors have determined the origin of and characterised this background amplification. This class of background amplification has been termed “AmpX”. The inventors have determined that it is an alternative amplification reaction which utilizes any linear nucleic acid probe molecules present in the reaction mixture. Typically the reaction products are AmpX multimers of head to tail tandem repeats. However, the inventors have determined that rather than encompassing sequence from the entire circular probe, the products of the AmpX reaction include repeats of a region of the linear target molecule that includes the two primer binding sites, the intervening sequence and some additional sequence of the template molecule flanking the primer binding sites.
Accordingly, the inventors have developed a method for minimizing AmpX background amplification by enriching for closed circular nucleic acid probe molecules prior to their amplification. By conducting the amplification step utilising an enriched population of closed circle nucleic acid probe molecules the incidence of background amplification caused by the AmpX reaction is significantly reduced, thereby enabling more specific rolling circle amplification to occur.